Invasive fungal infections, specifically candidemia, constitute major public health problems with high mortality rates. Therefore, in the last few years, the development of novel diagnostic methods has been considered a critical issue. Herein we describe a multiplex PCR strategy allowing the identification of 8 clinically relevant yeasts of the Candida genus, namely C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. guilliermondii, C. lusitaniae and C. dubliniensis. This method is based on the amplification of two fragments from the ITS1 and ITS2 regions by the combination of 2 yeast-specific and 8 species-specific primers in a single PCR reaction. Results from the identification of 231 clinical isolates are presented pointing to the high specificity of this procedure. Furthermore, several Candida isolates were identified directly from clinical specimens which also attests to the method's direct laboratory application. The results from the multiplex reactions with other microorganisms that usually co-infect patients also confirmed its high specificity in the identification of Candida species. Moreover, this method is simple and presents a sensitivity of approximately 2 cells per ml within 5 hours. Furthermore, it allows discrimination of individual Candida species within polyfungal samples. This novel method may therefore provide a clinical diagnostic procedure with direct applicability.Agostinho Carvalho was financially supported by a fellowship from Fundação para a Ciência e Tecnologia, Portugal (contract SFRH/BD/11837/2003). This study was supported by Fundação para a Ciência e Tecnologia, Portugal (POCI/SAU-ESP/61080/2004)
